Rutin alleviates bisphenol A-glycidyl methacrylate-induced generation of proinflammatory mediators through the MAPK and NF-κB pathways in macrophages.

Bisphenol A-glycidyl methacrylate (BisGMA) is a methacrylate monomer that is mainly used in three-dimensional structures to reconstruct dental and bony defects. BisGMA has toxic and proinflammatory effects on macrophages. Rutin is a natural flavonol glycoside that is present in various plants and has useful biological effects, such as anti-inflammatory, anticancer, and antioxidative effects. The aim of this study was to investigate the anti-inflammation of rutin in macrophages after exposure to BisGMA. Pretreatment of the RAW264.7 macrophage with rutin at 0, 10, 30, and 100 µM for 30 min before being incubated with BisGMA at 0 or 3 µM. Proinflammatory cytokines and prostaglandin (PG) E2 were detected by enzyme-linked immunosorbent assay (ELISA). Nitric oxide (NO) was detected by the Griess assay. Expression and phosphorylation of proteins were measured by Western blot assay. Pretreatment with rutin inhibited the BisGMA-induced generation of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and PGE2, in macrophages. Rutin also suppressed the BisGMA-induced secretion of NO and expression of inducible nitric oxide synthase (iNOS) in a concentration-dependent manner. Furthermore, rutin suppressed the mitogen-activated protein kinase (MAPK) phosphorylation in a concentration-dependent manner. Finally, rutin suppressed the BisGMA-induced phosphorylation of nuclear factor (NF)-κB p65 and degradation of inhibitor of κB (IκB). These results indicate that the concentration of rutin has an inhibitory effect on proinflammatory mediator generation, MAPK phosphorylation, NF-κB p65 phosphorylation, and IκB degradation. In conclusion, rutin is a potential anti-inflammatory agent for BisGMA-stimulated macrophages through NF-κB p65 phosphorylation and IκB degradation resulting from MAPK phosphorylation.

Investigating the in vitro metabolism of the dental resin monomers BisGMA, BisPMA, TCD-DI-HEA and UDMA using human liver microsomes and quadrupole time of flight mass spectrometry.

Dental resin systems have been in use for several decades. (Meth)acrylic monomers are an important part of the matrix system and are either based on BPA while others lack the BPA core. The degree of conversion during restoration is in general between 50-70 % allowing leaching from unreacted monomers to the oral cavity where they can be taken up through the pulp or gastrointestinal tract after ingestion with subsequent hepatic metabolism. This study identified the in vitro Phase I and Phase II metabolism of the dental resin monomers BisGMA, UDMA, BisPMA and TCD-DI-HEA, using human liver microsomes (HLM) and human liver cytosols. During Phase I incubation with HLM, the (meth)acrylic acid in the monomers was rapidly removed followed by oxidative and hydroxylation pathways. For BisPMA an O-dealkylation pathway occurred resulting in the formation of BPA. The carbamates present in TCD-DI-HEA and UDMA were resistant to biotransformation reactions. Phase II biotransformation products were only observed for BisPMA and included conjugation reactions with sulphate and glucuronic acid. In total 4, 3, 12 and 3 biotransformation products were identified in this study for BisGMA, UDMA, BisPMA and TCD-DI-HEA respectively. Possible human health effects of these biotransformation products remain unclear due to limited data availability.

Mechanistic, genomic and proteomic study on the effects of BisGMA-derived biodegradation product on cariogenic bacteria.

OBJECTIVES: Investigate the effects of a Bis-phenyl-glycidyl-dimethacrylate (BisGMA) biodegradation product, bishydroxypropoxyphenyl-propane (BisHPPP), on gene expression and protein synthesis of cariogenic bacteria. METHODS: Quantitative real-time polymerase chain reaction was used to investigate the effects of BisHPPP on the expression of specific virulence-associated genes, i.e. gtfB, gtfC, gbpB, comC, comD, comE and atpH in Streptococcus mutans UA159. Possible mechanisms for bacterial response to BisHPPP were explored using gene knock-out and associated complemented strains of the signal peptide encoding gene, comC. The effects of BisHPPP on global gene and protein expression was analyzed using microarray and quantitative proteomics. The role of BisHPPP in glucosyltransferase (GTF) enzyme activity of S. mutans biofilms was also measured. RESULTS: BisHPPP (0.01, 0.1mM) up-regulated gtfB/C, gbpB, comCDE, and atpH most pronounced in biofilms at cariogenic pH (5.5). The effects of BisHPPP on the constructed knock-out and complemented strains of comC from quorum-sensing system, implicated this signaling pathway in up-regulation of the virulence-associated genes. Microarray and proteomics identified BisHPPP-regulated genes and proteins involved in biofilm formation, carbohydrate transport, acid tolerance and stress-response. GTF activity was higher in BisHPPP-exposed biofilms when compared to no-BisHPPP conditions. SIGNIFICANCE: These findings provide insight into the genetic and physiological pathways and mechanisms that help explain S. mutans adaptation to restorative conditions that are conducive to increased secondary caries around resin composite restorations and may provide guidance to clinicians' decision on the selection of dental materials when considering the long term oral health of patients and the interactions of composite resins with oral bacteria.

Biodegradation of composite resin with ester linkages: identifying human salivary enzyme activity with a potential role in the esterolytic process.

OBJECTIVES: The ester linkages contained within dental resin monomers (such as Bisphenol A-glycidylmethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA)) are susceptible to hydrolytic degradation by salivary esterases, however very little is known about the specific esterase activities implicated in this process. The objective of this work was to isolate and identify the dominant proteins from saliva that are associated with the esterase activities shown to be involved in the degradation of BisGMA. METHODS: Human whole saliva was collected and processed prior to separation in a HiPrep 16/60 Sephacryl S-200 HR column. The fraction with the highest esterase activity was further separated by an anion exchange column (Mono-Q (10/100G)). Isolated fractions were then separated by gel electrophoresis, and compared to a common bench marker esterase, cholesterol esterase (CE), and commercial albumin which has been reported to express esterase activity. Proteins suspected of containing esterase activity were analyzed by Mass Spectroscopy (MS). Commercially available proteins, similar to the salivary esterase proteins identified by MS, were used to replicate the enzymatic complexes and confirm their degradation activity with respect to BisGMA. RESULTS: MS data suggested that the enzyme fraction with the highest esterase activity was contained among a group of proteins consisting of albumin, Zn-α2-glycoprotein, α-amylase, TALDO1 protein, transferrin, lipocalin2, and prolactin-induced protein. Studies concluded that the main esterase bands on the gels in each fraction did not overlap with CE activity, and that albumin activity emerged as a lead candidate with significant esterase activity relative to BisGMA degradation, particularly when it formed a complex with Zn-α2-glycoprotein, under slightly basic conditions. SIGNIFICANCE: These enzyme complexes can be used as a physiologically relevant formulation to test the biostability of composite resins.

Calcium phosphate glasses: silanation process and effect on the bioactivity behavior of glass-PMMA composites.

This article presents the results of a study of the efficiency of silanation process of calcium phosphate glasses particles and its effect on the bioactivity behavior of glass- poly(methyl methacrylate) (PMMA) composites. Two different calcium phosphate glasses: 44.5CaO-44.5P2 O5 -11Na2 O (BV11) and 44.5CaO-44.5P2 O5 -6Na2 O-5TiO2 (G5) were synthesized and treated with silane coupling agent. The glasses obtained were characterized by Microprobe and BET while the efficiency of silanation process was determined using Fourier Transform Infrared Spectroscopy (FTIR), X-ray Photoelectron Spectroscopy (XPS) and Thermal Analysis (DTA and TG) techniques. The content of coupling agent chemically tightly bond to the silanated glasses ascended to 1.69 ± 0.02 wt % for BV11sil glass and 0.93 ± 0.01 wt % for G5sil glass. The in vitro bioactivity test carried out in Simulated Body Fluid (SBF) revealed certain bioactive performance with the use of both silanated glasses in a 30% (by weight) as filler of the PMMA composites because of a superficial deposition of an apatite-like layer with low content of CO3 (2-) and HPO4 (2-) in its structure after soaking for 30 days occurred.

Effect of salivary esterase on the integrity and fracture toughness of the dentin-resin interface.

Human Salivary Derived Esterases (HSDE) are part of the salivary group of enzymes which show strong degradative activity toward the breakdown of one of the most common monomers used in dental composites and adhesives, 2,2-[4(2-hydroxy 3-methacryloxypropoxy)-phenyl] propane (Bis-GMA), to form the degradation product 2,2-bis [4 (2,3-hydroxy-propoxy)phenyl] propane (Bis-HPPP). This study was aimed to evaluate the effects of HSDE on the biodegradation and fracture toughness of the adhesive resin-dentin interface. Adhesive resin (Scotchbond Multi Purposes), resin composite (Z250) and mini short-rod specimens, were either not incubated; or incubated in phosphate-buffered saline (PBS) or HSDE media for up to 180 days (37 degrees C, pH 7.0). The amount of Bis-HPPP was analyzed by high performance liquid chromatography and mini-SR specimens were tested for fracture toughness using universal testing machine following 30, 90, or 180-day incubation periods. Significantly higher amounts of Bis-HPPP were produced in HSDE than in PBS incubated specimens (p < 0.05). Non-incubated mini-SR specimens had the higher fracture-toughness values, while specimens incubated for 180-days in HSDE had the lowest fracture toughness (p < 0.05). This study suggests that biodegradation is an on-going clinically relevant process that progressively compromises the integrity of the critical resin restoration-adhesive interface, as well as the resin-composite component with time.

In vitro enzymatic biodegradation of adhesive resin in the hybrid layer.

Penetration of adhesives into the demineralized dentin surface and their subsequent conversion are critically important to longevity of the adhesive resin (AR)-dentin bond. The durability of the resin-dentin bond is investigated by monitoring the change of adhesive concentration within the hybrid layer (HL) of aged specimens using Raman spectroscopy. Absolute molar concentrations of Bis-GMA and HEMA were measured across the HL of resin-dentin specimens 24 h after photopolymerization and after 24-week storage in one of three media: artificial saliva (SAL), SAL containing cholesterol esterase to attack resin (EST), and SAL containing bacterial collagenase to attack collagen (COL). No significant difference among these groups for both Bis-GMA and HEMA molar concentrations at 24-h storage was found; however, concentrations decreased from the AR to the middle of the HL. Concentrations remained unchanged at any resin-dentin position after aging in SAL. In the HL, concentrations significantly decreased with aging in COL and tended to decrease in EST. While showing potential enzymatic biodegradative effects of endogenous matrix metalloproteinases and salivary esterases, this methodology may also prove to be a valuable assessment of new chemistries and future approaches to improve resin-dentin bond performance. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Water sorption and diffusion coefficient through an experimental dental resin.

Polymeric composites have been widely used as dental restorative materials. A fundamental knowledge and understanding of the behavior of these materials in the oral cavity is essential to improve their properties and performance. In this paper we computed the data set of water absorption through an experimental dental resin blend using specimen discs of different thicknesses to estimate the diffusion coefficient. The resins were produced using Bisphenol A glycol dimethacrylate, Bisphenol A ethoxylated dimethacrylate and Triethylene glycol dimethacrylate monomers. The water sorption test method was based on International Standard ISO 4049 "Dentistry-Polymer-based filling materials". Results show a diffusion coefficient around 6.38 x 10(-8) cm(2)/s, within a variance of 0.01%, which is in good agreement with the values reported in the literature and represents a very suitable value.

Composite resin degradation products from BisGMA monomer modulate the expression of genes associated with biofilm formation and other virulence factors in Streptococcus mutans.

Bacterial microleakage along the tooth/composite resin dental restoration interface contributes to postoperative sensitivity, recurrent caries, and necrosis. Studies have confirmed that enzymes in human saliva degrade composite resin monomers 2,2-bis [4-(2-hydroxy-3-methacryloxypropoxy) phenyl] propane (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) to release methacrylic acid (MA), bishydroxypropoxyphenyl propane (Bis-HPPP), and triethylene glycol (TEG) at levels of 50 microM in vivo. Studies have found that TEGDMA degradation products alter the growth and gene expression of cariogenic Streptococcus mutans. Specifically, TEG was shown to alter S. mutans gene expression levels of gtfB, a known virulence factor, and yfiV, a putative transcriptional regulator of cell-surface fatty acid genes. The objective of this study was to examine the effect of BisGMA degradation products on the growth and gene expression of S. mutans NG8 cells. Results demonstrated slight inhibition of bacterial growth at Bis-HPPP concentrations of 1.0 x 10(2) and 2.5 x 10(2) microM at pH 5.5. Furthermore, both MA and Bis-HPPP affected gtfB and yfiV expression in a concentration-dependent manner. Because BisGMA is universally used across most dental restorative materials, with millions of placement procedures performed annually, these findings are relevant due to the potential influence of resin monomer-derived biodegradation products on biofilm formation, acid tolerance, and proliferation of S. mutans cells.

Effect of bacterial collagenase on resin-dentin bonds degradation.

The objective of this study is to evaluate the effect of a bacterial collagenase on the degradation of resin-dentin bonds. Human dentin surfaces were bonded with: an etch-&-rinse self-priming adhesive (SB), a two-step self-etching primer/adhesive (SEB), and a 1-step self-etching adhesive (OUB). Composite build-ups were constructed. The bonded teeth were stored (24 h, 3 months, 1 year) in distilled water or in a buffered bacterial collagenase solution. Half of the specimens were stored as intact bonded teeth (Indirect Exposure/IE). The other half were sectioned into beams prior to storage (Direct Exposure/DE). After storage the intact teeth were sectioned into beams and all specimens were tested for microtensile bond strengths (MTBS). ANOVA and multiple comparisons tests were performed. Fractographic analysis was performed by scanning electron microscopy. The inclusion of bacterial collagenase in the storing solution did not lower the MTBS values over those seen in specimens stored in water. SB and SEB bonds strength were equal, and were superior to OUB. After 3 months of DE, SB and OUB bonded specimens showed decreases in MTBS; similar reductions required 1 year for SEB/DE. MTBS did not decrease in IE specimens except for OUB. Resin and collagen dissolution were evident in DE groups after storing.

Differential effect of in vitro degradation on resin-dentin bonds produced by self-etch versus total-etch adhesives.

OBJECTIVE: To evaluate the effect of an in vitro challenge (NaOCl immersion) on microtensile bond strength (MTBS) of five adhesive systems to dentin. METHODS: Flat dentin surfaces from 40 molars were bonded with three total-etch adhesives (Single Bond, Prime&Bond NT and the experimental Prime&Bond XP), and two self-etching agents (Clearfil SE Bond and Etch&Prime 3.0). Composite build-ups were constructed with Tetric Ceram. Teeth were then sectioned into beams of 1.0 mm2 cross-sectional area. Half of the beams were immersed in 10% NaOCl aqueous solution for 5 h. Each beam was tested in tension in an Instron machine at 0.5 mm/min. Data were analyzed by 2-way ANOVA and multiple comparisons tests (p < 0.05). RESULTS: Clearfil SE Bond and Single Bond attained higher MTBS than the other three adhesives. Prime&Bond NT and Prime&Bond XP performed equally, and Etch&Prime resulted in the lowest MTBS. After NaOCl immersion, MTBS decreased in all groups. The highest MTBS values were obtained for Clearfil SE Bond and Prime&Bond XP. Scaning electron microscopy observation of debonded sticks evidenced dissolution and microstructural alterations of intertubular dentin, except when Clearfil SE Bond was used. CONCLUSIONS: Resin-dentin bonds are prone to chemical degradation. The extent of the resin degradation is adhesive system specific. Chemical degradation of the nonresin infiltrated collagen fibers does also exist in total-etch adhesives. Both processes may reduce long-term resin-dentin bond strength.

Cytotoxicity of bisphenol A glycidyl methacrylate on cytochrome P450-producing cells.

Of the cytochrome P450 (CYP) family of carcinogen-activating enzymes, CYP3A is the major form found in human livers. The purpose of this study was to investigate the cytotoxic effects of dental resin monomers after being metabolized by CYP3A4 and CYP3A7, using a colony formation assay and a neutral red assay. Specimen wells were plated with transfected cells derived from the Chinese hamster lung at 100 cells well(-1). The experimental group consisted of CYP-producing 3A4-10 and 3A7-40 cells, while the control group consisted of non-CYP-producing CR-119 cells. Bisphenol A (BPA) and bisphenol A glycidyl methacrylate (Bis-GMA) and a positive control (Aflatoxine Bl) were added separately to each well and cultured for 7 days. After cultivation, the number of the colonies was counted and IC50 values were determined. The data were statistically analysed by a Student's t-test. The resultant of IC50 values indicated that the monomers were not metabolically activated by CYP3A4 or CYP3A7 as compared with the control (P < 0.05). We also confirmed that these monomers act neither as activators nor as inhibitors of CYP3A4 and CYP3A7.

Estrogenicity of resin-based composites and sealants used in dentistry.

We tested some resin-based composites used in dentistry for their estrogenic activity. A sealant based on bisphenol-A diglycidylether methacrylate (bis-GMA) increased cell yields, progesterone receptor expression, and pS2 secretion in human estrogen-target, serum-sensitive MCF7 breast cancer cells. Estrogenicity was due to bisphenol-A and bisphenol-A dimethacrylate, monomers found in the base paste of the dental sealant and identified by mass spectrometry. Samples of saliva from 18 subjects treated with 50 mg of a bis-GMA-based sealant applied on their molars were collected 1 hr before and after treatment. Bisphenol-A (range 90-931 micrograms) was identified only in saliva collected during a 1-hr period after treatment. The use of bis-GMA-based resins in dentistry, and particularly the use of sealants in children, appears to contribute to human exposure to xenoestrogens.